The rapid advancement of gene therapy has intensified the demand for robust analytical techniques capable of ensuring the quality, safety, and efficacy of complex biotherapeutics. Among these, oligonucleotide-based drugs and adeno-associated virus (AAV) vectors represent two of the most promising modalities. This lecture explores state-of-the-art chromatographic strategies tailored to the characterization and quality control of these therapeutic platforms.

For oligonucleotide therapeutics, including antisense oligonucleotides, siRNAs, and aptamers, chromatographic methods [1-2] such as ion-pair reversed-phase liquid chromatography (IP-RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEX), and size exclusion chromatography (SEC) are employed to resolve sequence-related impurities, truncated species, and conjugates. These techniques are coupled with UV or mass spectrometric (MS) detection to enhance sensitivity and structure identification.

In the context of AAV gene therapy, chromatography plays a pivotal role in both upstream and downstream analytics [3]. Affinity chromatography (AFC) is widely used for capsid capture, while anion-exchange chromatography (AEC) and size-exclusion chromatography (SEC) enable separation of full and empty capsids, aggregate profiling, and impurity quantification. Coupling these methods with multi-angle light scattering (MALS) or LC-MS provides deeper insights into capsid integrity, payload content, and process-related contaminants such as residual iodixanol (gradient ultracentrifugation).

Together, these chromatographic approaches form a comprehensive analytical toolbox that supports the development, manufacturing, and regulatory compliance of oligonucleotide and AAV-based therapeutics. The lecture will highlight recent innovations, practical challenges, and case studies that illustrate the evolving landscape of analytical science in gene therapy.

References
[1] Y. Obata et al., J Chromatogr A, 2025, 1750: 465915
[2] MG. Bartlett, J Chromatogr A, 2024,1736: 465378
[3] S. Kurth et al., Analyt Biochem, 2024, 686: 115421

Cell therapy is a medical treatment that involves administering live cells into a patient to repair, replace, or enhance damaged tissues or cellular functions, offering potential cures for a wide range of diseases, including cancer, autoimmune disorders, and degenerative conditions. Despite its promise, the characterization and analysis of cell-based therapies present significant challenges that must be addressed to ensure their clinical success and widespread adoption.

A key challenge lies in developing robust analytical methods to assess the identity, potency, safety, and purity of cell therapy products, according to ICH guidelines [1] [2]. AGC Biologics has taken significant steps to overcome these hurdles by designing and optimizing a comprehensive panel of in-house tests for the characterization of the Drug Substance (DS) and the release of the final Drug Product (DP). Leveraging automation solutions, some of these tests have been automated to enhance precision, reduce release timelines and volume dedicated to analysis.

The inherent heterogeneity of cell populations, combined with process variability, further complicates the characterization process. Regulatory requirements add another layer of complexity, demanding detailed and reliable data on cell therapy products. Advanced technologies such as flow cytometry, droplets digital PCR, Rapid test have become indispensable tools in meeting these stringent demands.

In certain clinical scenarios, the need for rapid release and, in some cases, fresh infusion of cell therapy products introduces additional obstacles. Freshly prepared cell therapies often require immediate administration to preserve their viability and therapeutic potential, making traditional characterization timelines impractical. Balancing the urgency of delivery with the requirement for thorough characterization and quality assurance remains a critical issue. Innovative approaches, such as real-time analytics, predictive modelling, and streamlined release protocols, are essential to enable quick yet reliable processes without compromising patient safety.

References
[1] ICH Q2(R2) Guideline on validation of analytical procedures
[2] ICH Q14 Guideline on analytical procedure development

Oligonucleotide-based therapeutics and gene therapies are increasingly attracting attention due to advancements in their efficacy and the resolution of delivery-related challenges. Modalities such as antisense oligonucleotides (ASOs) are gaining prominence because of their exceptional specificity and their capacity to target previously undruggable molecular sites. To ensure the safety and efficacy of these therapeutics, robust methodologies for the precise identification and comprehensive characterization of the full-length product (FLP) and its associated impurities are paramount. High Resolution Accurate mass spectrometry (HR-MS) is instrumental for detecting potential impurities by comparing experimentally measured accurate masses and isotope patterns to theoretical values. However, the lack of sophisticated yet user-friendly data processing software hampers efficient analysis, necessitating labor-intensive and time-consuming manual interpretation. Additionally, structural confirmation through High Resolution tandem MS (HR-MS/MS) introduces another layer of analytical complexity.

The present study outlines the identification and relative quantification of oligonucleotide impurities using specific software to address common challenges associated with data analysis. Furthermore, previously undetected impurities in the FLP were identified. Impurity quantification, based on time-of-flight mass spectrometry (TOF MS) data, was performed within the software framework, and MS/MS fragmentation patterns of the impurities were utilized to confirm their molecular structures. This workflow facilitates the in-depth characterization of therapeutic candidates in development.

References
[1] Development of an ultra-sensitive assay for anti-sense oligonucleotide quantification. SCIEX technical note, RUO-MKT-02-13320-A.

Cell and gene therapies represent a broad pipeline of bioengineered treatments for disease, encompassing gene editing and de novo expression, hematopoietic stem cell transplantation and immune reconstitution, targeted immunotherapy, and tissue regeneration. As cell and gene therapies are becoming increasingly important for the treatment of cancers and other diseases, the need for reliable characterization of such therapies is skyrocketing [1].

Flow cytometry has emerged as a key methodology for the analysis of cell and gene therapies during research, drug development, clinical manufacturing and follow-up in patients. Flow cytometry is a technology that rapidly analyzes single cells or particles as they flow past single or multiple lasers while suspended in a buffered salt-based solution. Each particle is analyzed for visible light scatter and one or multiple fluorescence parameters. It allows for the simultaneous characterization of mixed populations of cells from blood and bone marrow as well as solid tissues that can be dissociated into single cells such as lymph nodes, spleen, mucosal tissues, solid tumors etc. [2]

Due to its potential applications and the impact on the results produced with flow cytometry assays, having robust and reliable validated assays is mandatory to allow the analysis of (pre)clinical samples. However, flow cytometry methods can be challenging to develop and validate due to several reasons: the inherent biological variability of cells, the appropriate selection and characterization of the reagents used and the lack of standardized cellular reference materials or quality controls, the limited stability of samples and the complexity of data output and interpretation of results [3,4]. Although there are no official regulatory guidance documents addressing the requirements for the validation of flow cytometric methods intended for use in drug development or for clinical testing, in the past few years, key stakeholders have published recommendation papers [4].

This lecture will present an overview on the use of flow cytometry and recommendations for method validation in accordance with regulatory requirements, based on the fit for purpose approach.

References
[1] Lazarski C.A. et al. Cytotherapy. 2024, 26(2),103-112
[2] McKinnon K.M. et al. Curr Protoc Immunol. 2018, 120, 5.1.1-5.1.11
[3] van der Strate B. et al. Bioanalysis, 2017, 9(16), 1253-1264.
[4] O Hara D.M. et al. Journal of Immunological Methods. 2011, 363 (2), 120-134

mRNA therapeutics, beyond vaccines, offer potential treatments for various diseases due to their safe, non-integrative, and transient cellular expression. Although manufacturing is straightforward, factors such as yield, scale, and purity impact costs and efficiency. Ideal mRNA therapeutics should be safe, effective, stable, and produced reliably and cost-effectively for on-demand use.

Drug developers require solutions to accelerate the development process while mitigating scaling and manufacturing risks. Monitoring key parameters such as yield, purity, and resolution is crucial in the production of plasmid DNA (pDNA) and mRNA. By utilizing a weak anion exchanging analytical column, significant improvements in yield and purity of plasmid pDNA were achieved. Rapid and reproducible separation and quantification of different isoforms and optimization of the pDNA was facilitated and accelerated. This enhancement significantly improved the overall quality and efficiency of pDNA production, which is crucial for generating high-quality mRNA. Further implementation of a multimodal analytical column in the mRNA production process played a crucial role in optimizing the in vitro transcription (IVT) reaction. By closely monitoring nucleoside triphosphates (NTPs) consumption and quantifying the transcribed RNA, it facilitated the transition from batch to fed-batch process, thereby not only boosting yield but also reducing overall production costs. Monitoring RNA integrity is essential to ensure the final product purity and consistency. To enhance the overall purification process and quality control, a method based on an ion-pairing reverse-phase analytical column was developed, which facilitated the separation of RNA fragments by size, allowing for the precise tracking of mRNA integrity and the removal of impurities like DNA and double-stranded RNA. Additionally, the column has proven reliable in stability tests, aligning well with traditional methods like capillary gel electrophoresis, and confirming that mRNA samples remained intact under various storage conditions. By utilizing these advanced tools, researchers and manufacturers can gain critical insights into the synthesis process, achieve enhanced purity, and improve resolution in mRNA production, ultimately contributing to more reliable and effective mRNA-based treatments.

References
[1] M. Leban, T. Vodopivec Seravalli, M. Hauer. et al., Anal. Bioanal. Chem.,2024, 416, 2389-2398
[2] P. Megusar, E.D.D Calder, T. Vodopivec Seravalli, S. Lebar, L.J. Walport, R. Sekirnik, 2024, Front. Mol. Biosci., 11:1443917.
[3] A. Ferjanxix Budihna, A. Martincic Celjar, S. Lebar, A. Gramc Livk, A. Strancar, Cell gene ther. Insights, 2023, 9(9), 1231-1247
[4] N. Pavlin, T. Kovaxix, T. Plesnicar, A. Gramc Livk, A. Strancar, Cell gene ther. Insights, 2024, 10(6), 867-878

Advanced Therapy Medicinal Products (ATMPs) form a new class of biological drugs that can be used to treat unmet medical conditions and go one step further the personalized medicine. One of the main concerns about the use of ATMPs is the safety of patients because ATMPs can induce an immunological response with a consequent impact on treatment efficacy rather than patients impairment.

So far, to put the new therapy on the market, a regulated risk-based approach for ATMPs development is strongly suggested [1] and, as per guideline, should include the immunogenicity evaluation starting from the non-clinical studies as part of the overall toxicology assessment [2].

ATMPs can elicit an immune response involving the innate or the adaptive immune systems. The difference of such activation is linked to the fact that ATMPs are delivered for the first time in the organism and are recognized as foreign or they are identified similar to a previously encountered substance.

Different bioanalytical methods can be used to monitor the immune response of ATMPs administered in vivo and the majority is represented by ligand binding assays (LBAs) such as ELISA. Each ATMP has unique properties which need to be considered when assessing its immunogenicity using LBAs.

A special focus on immunogenicity of gene therapy medicinal products (AAV, oligonucleotides) and cell-based products (CAR-T) will be provided with highlights to the LBA which can be used for the assessment and differences and similarities between non-clinical and clinical studies.

Adeno-associated viruses (AAVs) are icosahedral capsids comprised of 60 total copies of three viral proteins (VPs), VP1, VP2, and VP3, in an approximate 1:1:10 ratio, respectively. VP3 is the smallest of the three VPs, with VP2 containing the entire sequence of VP3 as the C-terminal sequence and VP1 containing the entirety of VP2 as its C-terminal sequence. These capsids contain single-strand (ss) DNA of approximately 4.7 kb that delivers the genetic payload to target cells. There is increasing interest in AAVs as gene therapy vectors because of their highly effective delivery mechanisms, low cytotoxicity, and minimal immunogenicity. Additionally, the variety of serotypes, each having different tropisms, provides the ability to target specific cell types and organs. Currently a handful of AAV therapy products are on the market, receiving conditional or full approval by either the US Food and Drug Administration (USFDA) or the European Medicines Agency (EMA). Monitoring AAV vector quality is crucial for ensuring product safety and efficacy. A key aspect of this is monitoring changes in post-translational modifications (PTMs) on the capsid VPs. PTMs on said VPs are known to occur during production and storage and can have an influence on product stability, infectivity, and transduction efficiency. They also are seen varying between production lots highlighting the importance of batch-to-batch monitoring. Additionally, final product yields for full AAV capsids are low due to the high levels of empty or partially filled capsids that are generated during production and need to be removed downstream. Therefore, having highly sensitive AAV characterization platforms is critical to minimize the amount of sample needed for quality control (QC) testing.

In this talk, several mass spectrometry based workflows for the analysis of different quality attributes will be explored. The talk will start from the monitoring of the full capsid filling states through advanced techniques such as charge detection mass spectrometry (CDMS) enabling characterization of complexes with a mass range between 2 and 10 MDa. We will then move to the viral protein level, to determine post-translational modifications at intact and peptide level. Final topic of the talk will cover the detection and quantitation through mass spectrometry techniques of the host cell contaminants derived by the process and how the purification strategies can impact their presence in the final product.

References
1. Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021;6(1):53.
2. Liu AP, Patel SK, Xing T, Yan Y, Wang S, Li N. Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry. Journal of pharmaceutical and biomedical analysis. 2020;189:113481.
3. Strasser L, Morgan TE, Guapo F, F�ssl F, Forsey D, Anderson I, Bones J. A Native Mass Spectrometry-Based Assay for Rapid Assessment of the Empty:Full Capsid Ratio in Adeno-Associated Virus Gene Therapy Products. Anal Chem. 2021 Sep 28;93(38):12817-12821.
4. Smith J, Carillo S, Kulkarni A, Redman E, Yu K, Bones J. Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS. Anal Bioanal Chem. 2024 Feb;416(4):1069-1084.
5. Smith J, Strasser L, Guapo F, Milian SG, Snyder RO, Bones J. SP3-based host cell protein monitoring in AAV-based gene therapy products using LC-MS/MS. Eur J Pharm Biopharm. 2023 Aug;189:276-280.

Real-Time Polymerase Chain Reaction, also called PCR, is a very powerful and sensitive bioanalysis technique. It is used for a broad range of applications such as quantitative gene expression analysis, biodistribution studies of a nucleic acid target sequence, genotyping, single nucleotide polymorphisms (SNP) analysis, pathogen detection, drug target validation and for measuring RNA interference. The quantitative PCR (qPCR) method is used for the detection and quantitation of an amplified PCR product as the reaction progresses and is based on the incorporation of a fluorescent dye. qPCR can be combined with reverse transcription (rt) assay in case of RNA as starting material in cells or tissues in two separated analysis or in one-step reaction (rt-qPCR). This presentation is focused on the experimental activities and procedures required for the validation of the bioanalytical methods used to quantify by PCR a specific nucleic acid in support of Biodistribution studies. Biodistribution studies are required for the development of Gene Therapies (GT) and Cell Therapies (CT) modalities, since based on nucleic acids (DNA or RNA). The aim of these studies is to characterize the distribution and persistence of a gene/cell therapy product from the site of administration to target and non-target tissues and biofluids by measuring the vector genome or expression of the transgene, as well as drug-derived gene products. They are applied, to an extent defined by a careful risk-assessment evaluation agreed with the project owner and with the regulatory agencies if needed, to non-clinical and clinical studies that are conducted in support of regulatory applications and subjected to GLP/GCP requirements. To date, there has not been a regulatory guidance document with a descriptive approach on the validation of molecular assays that are used in regulated bioanalysis to support these novel modalities. Therefore, an internal evaluation was performed starting from available references [1] [2], to define the strategy to adopt for qPCR Method Validation for Biodistribution and Viral Shedding Studies and Sample Analysis. The procedure described consists in three different steps: PCR assay set-up and preliminary assessments that is part of method development and shall not constitute part of the validation study, validation of the qPCR reaction with the specific primers and probe set and validation of the qPCR process starting from biological samples. The same process applies for RT-qPCR assay. Each of these phases will be further detailed in each of the critical parts.

References
[1] A. Hays, et al., The AAPS Journal., 2024, 26:24.
[2] A. Lauren, et al., Bioanalysis , 2022, 14(16), 1085-1093.

Oligonucleotide drugs have increasingly gained attention and the disease areas for which these therapeutic agents are evaluated have been rapidly expanding. Oligonucleotides encompass a wide array of chemistries and modes of action, e.g. from single stranded antisense oligonucleotides to double stranded conjugated siRNAs. To determine their PK/PD properties, sensitive and selective quantitative bioanalytical methods are required. LC-MS has emerged as a powerful tool and one of the most selective bioanalytical technologies to quantify a variety of oligonucleotide therapeutics in biological matrices. Compared with alternative assay types (e.g. hybridization-ELISA), LC-MS methods can selectively quantify the full-length oligonucleotides and their major metabolites, have a much wider dynamic range and do not require any specific reagents. However, significant challenges should be addressed while developing an LC-MS assay for oligonucleotides, including their very high plasma protein binding, nonspecific binding to containers, lack of chromatographic retention, conjugation (e.g. with lipids) affecting the extractability and chromatographic separation, multiple charge states and formation of cation adducts affecting the LC-MS method performance. The sensitivity of LC-MS methods for quantification of oligonucleotides has significantly improved since the last few years and reaching sub ng/mL lower limits of quantification is now feasible. This is mainly due to improved MS equipment, consolidated extraction techniques and the adoption of state-of-the-art chromatographic technologies. The LC-MS sensitivity is usually sufficient to support preclinical studies, particularly for analysis of tissues where these drugs exhibit their efficacy and are present at high concentrations. However, the sensitivity might not be sufficient to support clinical studies where the administered doses and the observed exposures are expected to be lower. Sensitivity will be also challenging in certain matrices, e.g. in plasma or serum when the administration of the oligonucleotide drug is not systemic. In all cases defined above, switching technology (e.g. LC-MS to hELISA) in order to meet a sensitivity target should be carefully evaluated because the selectivity of these assays can be different, and a direct comparison of the exposures obtained may not be possible.

According to the MIST guidance, the metabolites of oligonucleotide drugs should be quantified in case these significantly contribute to the pharmacological and toxicological effect. Again, the use of LC-MS offers significant advantages compared to other technologies due to the increased selectivity. However, additional complexity is because the metabolic profiles of oligonucleotides in plasma and in tissues are typically very different, resulting in different sensitivity requirements.

Considering all the above considerations and challenges, a proposed strategy is to generate preliminary metabolite i.d. information in plasma and tissues before a quantitative technology is selected and bioanalytical methods are fully validated to support regulatory toxicological studies. The pharmacological activities of predictable shortmers should also be evaluated to determine what analytes require quantification. The selectivity of LC-MS and hELISA assays vs. shortmers should also be assessed to select the technology to be fully validated for quantification.

All factors above will be discussed in this presentation, which includes an overview of recent advances in LC-MS bioanalysis of oligonucleotides, along with examples showing how different experimental conditions will affect the performance of the assay. Case studies will also be presented showing how the preliminary metabolite information and a comparison of selectivity/sensitivity of different technologies will help the bioanalytical team implement a proper analytical strategy in order to avoid complex bridging studies at later stages of drug development.

Messenger RNA (mRNA) or adenoviral vector (AdV) vaccines have gained unprecedented success during the COVID-19 pandemic, thus a comprehensive evaluation of the induced immunological response was essential for these types of vaccines [1].

This presentation outlines the current understanding of immune responses to COVID-19 vaccines, focusing on both humoral and cell-mediated immunity. Different techniques are described for the immune response analysis. The analysis is contextualized by the evolving epidemiology of SARS-CoV-2, including the emergence of variants.

The humoral immune response is primarily characterized by the production of anti-SARS-CoV-2 antibodies. The kinetics of these antibodies and their avidity evolve with vaccine doses [2]. The immune response can be measured using techniques such as chemiluminescent microparticle immunoassay (CMIA), microneutralization assays, and Plaque Reduction Neutralization Tests (PRNT) conducted under BSL3 conditions. Soluble markers such as cytokines and chemokines can be quantified using Luminex multiplex technology, which helps identify early innate immune signatures correlated with vaccine efficacy.

The cell-mediated immune response involves immunophenotyping of lymphocyte subpopulations using flow cytometry to assess T cell differentiation, maturation, and exhaustion [3]. Specific assays such as ELISPOT and FLUOROSPOT can be employed to quantify T cell activation, and intracellular cytokine staining provides functional characterization. Antibody-dependent cellular cytotoxicity (ADCC) is evaluated through fluorescence and luminescence-based in vitro assays against both the spike and nucleocapsid proteins of SARS-CoV-2. These assays demonstrated the role of cytotoxic responses in vaccinated individuals and in response to Omicron subvariants.

The data support the necessity of integrated immunological assays for the comprehensive evaluation of vaccine-induced protection, particularly in fragile populations.

References
[1] Teijaro, J.R., Farber, D.L. COVID-19 vaccines: modes of immune activation and future challenges. Nat Rev Immunol, 2021, 21, 195-197.
[2] Bullock, J.L., Jr.; Hickey, T.E.; Kemp, T.J.; Metz, J.; Loftus, S.; Haynesworth, K.; Castro, N.; Luke, B.T.; Lowy, D.R.; Pinto, L.A. Longitudinal Assessment of BNT162b2- and mRNA-1273-Induced Anti-SARS-CoV-2 Spike IgG Levels and Avidity Following Three Doses of Vaccination. Vaccines, 2024, 12, 516.
[3] Cui, Z., Luo, W., Chen, R. et al. Comparing T- and B-cell responses to COVID-19 vaccines across varied immune backgrounds. Sig Transduct Target Ther, 2023, 8, 179.

When manufacturing a drug, one of the important aspects is its metabolism in the human body. Studying the fate of a drug in the body is particularly important when evaluating the effectiveness of a therapy. This step in drug development can be particularly challenging, especially for antisense oligonucleotides, which are becoming increasingly important in the treatment of various diseases. They are synthetic, chemically modified compounds used to treat various diseases, mainly genetic ones, e.g., spinal muscular atrophy (SMA). The Spinraza drug with antisense oligonucleotide (nusinersen) is used to treat this disease. The structure of nusinersen is modified with 2�-O-2-methoxyethyl and phosphorothioate groups, which increase its stability in the body, since the activity of antisense oligonucleotides includes binding to complementary sequences of the targeted ribonucleic acids in cells. The application of these compounds as therapeutic agents has enforced the need for their purification, as well as the separation and identification of metabolites and impurities. Metabolites of antisense oligonucleotides are formed by the activity of enzymes, mainly exonucleases. They are typically the products of single-nucleotide deletion. The challenges in characterizing metabolites of any therapeutic oligonucleotides arise from their large molecular masses, complex structures, and high affinity to proteins. As a result, fully elucidating their structure is a challenge, and the development of effective analytical methods is crucial to enable their isolation, separation, and qualification.

This study aimed to develop a method for extracting and analyzing nusinersen and its metabolites from serum and cerebrospinal fluid samples obtained from patients undergoing treatment with Spinraza.

The research was funded by the National Science Centre (Cracow, Poland) under the Opus project (2023/51/B/NZ7/00537).

The development of cell and gene therapies represents one of the most transformative frontiers in modern medicine, offering unprecedented potential to treat, modify, or even cure diseases once considered intractable. This journey - from conceptualization to bedside application - is inherently complex, involving translational science, regulatory navigation, bioethical considerations, and the construction of new manufacturing and delivery paradigms. The process begins with a concept followed by a preclinical discovery, where candidate cells and genes are identified and engineered for therapeutic potential. This is followed by the design of clinical-grade protocols, Good Manufacturing Practice (GMP) compliance, and phased clinical trials to assess safety, efficacy, and hopefully to consistent long-term outcomes.

Within this dynamic landscape, we have been developing cell and gene therapies for a variety of medical applications. Our work emphasized the need for a structured, evidence-based approach to developing cell and gene-based therapeutics, while advocating for global standards and ethical commercialization.

Here we will be underscoring the paradigm shift in how cell-based interventions - such as mesenchymal stromal/stem cells (MSCs) and chimeric antigen receptor (CAR) T cells - are transitioning from experimental protocols to regulated medicinal products. We will highlight the logistical and scientific hurdles in scaling such therapies, including potency assays, standardization of cell sourcing, and ensuring product consistency across batches.

The translation of cell and gene therapies from concept to patient care requires a convergence of scientific innovation, manufacturing infrastructure, ethical foresight, and policy reform. This lecture aims to provide a roadmap for navigating this path-advocating not only for scientific rigor but also for patient-centered, responsible advancement. As the field continues to evolve, such these approaches will be essential in shaping a future where advanced therapies are safe, accessible, and transformative across a wide spectrum of diseases.

References
1) Omarini C, Catani V, Mastrolia I, Toss A, Banchelli F, Isca C, Medici D, Ponzoni O, Brucale M, Valle F, Baschieri MC, D'Amico R, Masciale V, Chiavelli C, Caggia F, Bortolotti CA, Piacentini F, Dominici M. Extracellular vesicles-derived miR-21 as a biomarker for early diagnosis and tumor activity in breast cancer subtypes. Biomark Res. 2025 Jan 23;13(1):14. doi: 10.1186/s40364-025-00724-y.
2) Grisendi G, Dall'Ora M, Casari G, Spattini G, Farshchian M, Melandri A, Masicale V, Lepore F, Banchelli F, Costantini RC, D'Esposito A, Chiavelli C, Spano C, Spallanzani A, Petrachi T, Veronesi E, Ferracin M, Roncarati R, Vinet J, Magistri P, Catellani B, Candini O, Marra C, Eccher A, Bonetti LR, Horwtiz EM, Di Benedetto F, Dominici M. Combining gemcitabine and MSC delivering soluble TRAIL to target pancreatic adenocarcinoma and its stroma. Cell Reports Medicine 2024 Aug 20;5(8):101685.
3) Chiavelli C, Prapa M, Rovesti G, Silingardi M, Neri G, Pugliese G, Trudu L, Dall'Ora M, Golinelli G, Grisendi G, Vinet J, Bestagno M, Spano C, Papapietro RV, Depenni R, Di Emidio K, Pasetto A, Nascimento Silva D, Feletti A, Berlucchi S, Iaccarino C, Pavesi G, Dominici M. Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma. Nature PJ Precis Oncol. 2024 Feb 1;8(1):26.
4) Golinelli G, Grisendi G, Dall'Ora M, Casari G, Spano C, Talami R, Banchelli F, Prapa M, Chiavelli C, Rossignoli F, Candini O, D'Amico R, Nasi M, Cossarizza A, Casarini L, Dominici M. Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma. Transl Oncol. 2022 Jan;15(1):101240. doi: 10.1016/j.tranon.2021.101240.

Advanced Therapy Medicinal Products (ATMPs) represent a significant breakthrough in modern medicine, offering new hope for conditions once considered untreatable - from rare diseases to cancer and more prevalent disorders. These innovative therapies form a diverse and rapidly evolving class of medicines, driven by continuous scientific advancements. While each ATMP holds the potential to transform patient care, they also present unique opportunities and challenges.

The development of ATMPs in Europe is governed by a highly complex and multilayered regulatory framework that ensures product safety, efficacy, and quality while balancing scientific innovation and patient access. ATMPs - including gene therapies, somatic-cell therapies, and tissue-engineered products - are primarily regulated under Regulation (EC) No 1394/2007 [1], which supplements the broader Directive 2001/83/EC governing medicinal products. The European Medicines Agency (EMA) and its Committee for Advanced Therapies (CAT) play a central role in scientific evaluation and marketing authorization.

However, ATMP development often intersects with additional regulatory regimes. For cell- and tissue-based products, the Substances of Human Origin (SoHO) framework - including Directive 2004/23/EC [2] - sets requirements for donation, procurement, and testing, ensuring traceability and donor protection. This adds an additional compliance layer, especially during early manufacturing stages. For ATMPs administered via or in combination with medical devices (e.g., cell-seeded scaffolds or gene delivery systems), the Medical Device Regulation (MDR, 2017/745) [3] and the In Vitro Diagnostic Regulation (IVDR, 2017/746) [4] introduce further obligations, including conformity assessments and clinical evaluation requirements. Determining the primary mode of action becomes critical for classification and regulatory pathway selection. Additionally, ATMPs involving genetically modified organisms (GMOs) - such as CAR-T cells or gene-edited stem cells - must adhere to Directive 2001/18/EC (deliberate release of GMOs) [5] and Directive 2009/41/EC (contained use of GMOs) [6]. This dual oversight can complicate clinical trial applications, as GMO-specific environmental risk assessments are required alongside clinical safety data, with national-level variations adding complexity.

Navigating these interconnected regulatory layers demands a holistic approach, early regulatory engagement, and strategic planning. Understanding the interplay of medicinal product, SoHO, device, and GMO frameworks is essential for successful ATMP development, ensuring both regulatory compliance and patient-centric innovation.

References
[1] Regulation (EC) No 1394/2007 of the European Parliament and of the Council of 13 November 2007 on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004.
[2] Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells.
[3] Medical Device Regulation (MDR, 2017/745) of the European Parliament and of the Council of 5 April 2017 on medical devices.
[4] In Vitro Diagnostic Regulation (IVDR, 2017/746) f the European Parliament and of the Council of 5 April 2017 on in vitro diagnostic medical.
[5] Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms.
[6] Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the contained use of genetically modified micro-organisms

ATMPs are Advanced Therapy Medicinal Products including gene therapy, somatic cell therapy, and tissue-engineered products. Often the development of non-conventional analytical methods is required, according to ICH guidelines [1, 2].

Developing analytical methods for ATMPs involves several key steps: 1. Understanding the Product: First, it is essential to have a thorough understanding of the ATMP, including its composition, mechanism of action, and the specific characteristics that need to be analyzed; 2. Defining Analytical Objectives: Determine what needs to be measured, such as potency, purity, identity, and safety. This will guide the selection of appropriate analytical techniques; 3. Defining the application: often the same analytical method can be applied from pre-clinical safety studies to DP characterization and also for the analysis of clinical trial samples; 4. Method Development and Optimization: This involves refining the chosen techniques to ensure they are sensitive, specific, reproducible, and robust; 5. Validation: Once developed, the methods must be validated according to regulatory guidelines to ensure they are reliable and can be consistently applied; 6. Continuous Improvement: As more data is gathered and technologies advance, analytical methods may need to be revisited and improved.

The design of the validation strategy should consider all the applications of the method, to save time and reduce cost and to optimize the use of precious materials required for validation.

References
[1] EMA/CHMP/ICH/172948/2019 ICH guideline M10 on bioanalytical method validation and study sample analysis, 2023
[2] ICH guideline Q2(R2) on validation of analytical procedures, 31 July2022